1000 genom projesinde yer alan CLOCK geni tek nükleotid polimorfizmlerinin in silico ve in vitro analizleri

Circadian rhythm is an internal process regulating ~24-h physiological and behavioral processes in organisms. In mammals, circadian rhythm is generated by transcription and translational feedback loop (TTFL) mechanism as a result of the interaction between core clock proteins. In TTFL, CLOCK and BMAL1 interact with each other and form a heterodimer to bind E-box sequences within the promoter region to initiate the transcription of the clock-controlled genes, including Period (Per) and Cryptochrome (Cry). Within the time, CRYs and PERs accumulate in the cytosol and then translocate into the nucleus with Casein Kinase Iε and repress BMAL1: CLOCK driven transcription. There are other auxiliary TTFLs exist that control circadian rhythm. Genetics and epidemiolocal studies suggest factors that disturb circadian rhythm result in susceptibility or may directly cause several diseases such as obesity, diabetes, cardiovascular diseases, aging, cancer, mood, and sleep disorders. Several single nucleotide polymorphisms (SNPs) for core clock genes have been identified and shown to be associated with different types of diseases. However, whether these SNPs contribute to the different type of the disease are ill-defined. One of the challenges in these approaches is that genome-wide association sequences (GWAS) studies have limitations in functional prediction. To address that, I developed using in vitro studies following the in-silico techniques to identify and characterize functional CLOCK SNPs from 1000 Genomes Ensemble to show the effect of a particular missense mutation on CLOCK protein on function. Such systematic approaches would allow us to discover SNPs with pathological effects and understand how these SNPs affect proteins' function. In this thesis, I performed a functional characterization of rare CLOCK missense variations (p.Phe104Cys, p.Leu118Arg, p.Asp119Val, p.Gly120Val, and p.Phe121Cys) identified from the Ensembl database. I initially analyzed these variations using computational tools. Results revealed that variants are located on the functionally important region of CLOCK. I used in vitro experimental approach and showed p.Leu118Arg, p.Asp119Val, and p.Phe121Cys CLOCK had reduced transactivation activity while p.Gly120Val CLOCK had increased transactivation along with BMAL1. However, p.Phe104Cys CLOCK had not been comparable transcriptional activity. To attrubitue these functional difference on the affinity between CLOCK SNPs and BMAL1, I performed co-immunoprecipitation between them Results indicated that p.Leu118Arg, p. Asp119Val, p.Gly120Val CLOCKs had reduced affinity to BMAL1and interestingly p.Phe121Cys CLOCK had increased the affinity to BMAL1. To gain more insight I further showed that the CRY1 had reduced repressor activity on p.Leu118Arg and p.Phe121Cys CLOCKs. Meanwhile, the estimation binding energy analysis of MD simulations supported the biochemical results, and binding energy analysis per residues was used to examine the mechanism of such SNPs effects. Collectively, I discovered that even single nucleotide changes in CLOCK directly affect the CLOCK functions. Hence, illumination of the effects of CLOCK SNPs would also help develop novel treatment strategies for diseases related to clock disruption for further studies and provide valuable information for the structure-function of CLOCK in circadian clock mechanism.